1. Field of the Invention
The invention relates to an amino acid sequence useful for stabilization of otherwise unstable proteins, particularly proteolytically sensitive proteins. The invention also relates to methods of producing stabilized proteins by direct attachment of the stabilizing sequence or through fusion proteins expressed from recombinant host cells.
2. Description of Related Art
One of the major problems in the use and preparation of therapeutic and commercial proteins is degradation by cell proteases. This may occur in vivo in plasma or inside a host cell when recombinant methods of polypeptide production are employed. Degradation plays an obvious role in the elimination of damaged or abnormal proteins but also affects half lives of normal proteins. Individual protein turnover rates may vary 1000 fold, depending on environmental and structural factors.
Two general factors contribute to the half-life of any given protein. One factor includes "global" features such as large size, hydrophobicity, thermal instability and charge characteristics. The other factor is related to sequence specific parameters of particular .alpha.-amino terminii, asparagine residues and so-called PEST sequences. Although correlations have been made between particular features and susceptibility to degradation, explicit degradation signals and pathways are not well defined. It is not even known whether peptide bond cleavage or biochemical marking is the primary event signaling protein destruction.
In contrast to degradation factors, there appear to be stabilization factors that enhance stability of a protein toward proteolysis. It has been suggested that both thermodynamic stability and proteolytic susceptibility are major determining factors in rate of degradation (Parsell and Sauer, 1989). There has been some evidence that particular sequences at the N-terminus of lambda repressor in Escherichia coli aid in maintaining a stable tertiary structure and therefore slow proteolytic degradation (Parsell and Sauer, 1989). Some evidence also suggests that the increased stability of certain fusion proteins may derive from the ability of a stable fusion partner such as protein G or .beta.-galactosidase to stabilize the tertiary structure of a proteolytically sensitive protein (Hellebust et al., 1989).
A particular amino acid sequence attached as a "tail" to the C-terminal of Arc, a small dimeric DNA-binding protein found in bacteriophage P22, has been found to stabilize that protein against proteolysis. The primary sequence of the tail consists of 25 amino acids which when attached to a heterologous protein, the LP57 mutant of .lambda.-repressor in Escherichia coli, also stabilized that protein (Bowie and Sauer, 1989).
Nevertheless, up until now, an amino acid sequence attachable to a proteolytically sensitive protein has not been found which will generally increase stability of these proteins. The value of such a sequence would lie in its ability to increase the half-life of commercially desired proteins for production in recombinant cell hosts and the potential to protect proteinaceous vaccines from serum protease degradation when injected directly into an animal.